Differential Effects of 3rd Trimester-Equivalent Binge Ethanol and Tobacco-Specific Nitrosamine Ketone Exposures on Brain Insulin Signaling in Adolescence.

Background
Fetal alcohol spectrum disorder (FASD) is associated with impairments in insulin and insulin-like growth factor (IGF) signaling through Akt pathways and altered expression of neuro-glial proteins needed for structural and functional integrity of the brain. However, alcohol abuse correlates with smoking, and tobacco smoke contains 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which like other nitrosamines, impairs insulin and IGF signaling.


Hypothesis
NNK exposure can serve as a co-factor in mediating long-term neuro-developmental abnormalities associated with FASD.


Design
Long Evans rat pups were IP administered ethanol (2 g/kg) on postnatal days (P) 2, 4, 6 and/or NNK (2 mg/kg) on P3, P5, and P7, simulating third trimester human exposures. Temporal lobes from P30 rats (young adolescent) were used to measure signaling through the insulin/IGF-1/Akt pathways by multiplex ELISAs, and expression of neuroglial proteins by duplex ELISAs.


Results
Ethanol, NNK, and ethanol + NNK exposures significantly inhibited insulin receptor tyrosine phosphorylation, and IRS-1 and myelin-associated glycoprotein expression. However, the major long-term adverse effects on Akt pathway downstream signaling and its targeted proteins including choline acetyltransferase, Tau, pTau, ubiquitin, and aspartate-β-hydroxylase were due to NNK rather than ethanol.


Conclusion
Alcohol and tobacco exposures can both contribute to long-term brain abnormalities currently regarded fetal ethanol effects. However, the findings suggest that many of the adverse effects on brain function are attributable to smoking, including impairments in signaling through survival and metabolic pathways, and altered expression of genes that regulate myelin synthesis, maturation and integrity and synaptic plasticity. Therefore, public health measures should address both substances of abuse to prevent "FASD".

Variability in the nature and severity of alcohol-related neurodegeneration suggests that cofactors may be critical to disease pathogenesis. In this regard, it is noteworthy that a very high percentage of heavy drinkers (up to 80%) also abuse tobacco products, typically by smoking cigarettes [18]. Although the overwhelming interest in studying adverse effects of alcohol-tobacco dual exposures has focused on carcinogenesis [19][20][21][22], particularly in relation to the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone (NNK) and its metabolites [20.23], previous studies demonstrated that limited, sub-mutagenic exposures to other nitrosamines, i.e. streptozotocinor N-nitrosodiethylamine (NDEA), cause brain insulin resistance, increased DNA damage, lipid peroxidation, mitochondrial dysfunction, ER stress, and impaired signaling through PI3K-Akt (24)(25)(26) and can exacerbate effects of ethanol [27]. The present study tests the hypothesis that submutagenic exposures to NNK are sufficient to cause neurodegeneration and possibly exacerbate the adverse effects of alcohol with respect to brain insulin/IGF resistance, oxidative stress, neuroglial gene expression, and myelin maintenance. NNK rather than tobacco smoke effects were studied because smoking could confound the results by causing pulmonary disease [28,29].

In vivo Model
Long Evans rat pups were divided into four groups and administered 50 μl IP injections of: saline vehicle as control; pharmaceutical grade ethanol (2g/kg in saline); NNK (2 mg/kg in saline); and ethanol + NNK. Ethanol treatments (binge) were administered on postnatal days (P) 2, 4, 6, and 8 [30][31][32], and NNK was administered on P3, P5, P7, and P9. These models simulated 3 rd trimester-equivalent human pregnancy exposures to alcohol and/or tobacco toxins. The rats were sacrificed at six weeks of age to examine long-term effects on temporal lobe insulin/IGF-1 signaling through Akt growth and metabolic pathways during late adolescence. All experiments were performed in accordance with protocols approved by Institutional Animal Care and Use Committee at the Lifespan-Rhode Island Hospital, and they conformed to guidelines established by the National Institutes of Health.

Statistics
For competitive, duplex, and multiplex ELISAS, each experimental group included 8-10 rats. Inter-group comparisons were made using two-way analysis of variance (ANOVA) with Tukey post hoc tests (GraphPad Prism 6, San Diego, CA). F-ratios and P-values are tabulated. Significant post hoc test differences and trends (0.05 < P < 0.10) are shown in the graphs.

Materials
Pharmaceutical grade ethanol was used in the in vivo experiments. The A85G6 and A85E6 monoclonal antibodies to ASPH were generated to human recombinant protein [34] and purified over Protein G columns (Healthcare, Piscataway, NJ). Otherwise, antibodies used for duplex ELISAs were purchased from Abcam (Cambridge, MA). RPLPO) antibody was from the Proteintech Group Inc (Chicago, IL). ELISA MaxiSorp 96-well plates were purchased from Nunc (Rochester, NY). Horseradish peroxidase (HRP)-conjugated secondary antibody, Amplex Red soluble fluorophore, and the Akt Pathway Total and Phospho panels were purchased from Invitrogen (Carlsbad, CA). HRP-labeled polymer conjugated secondary antibody was purchased from Dako Corp (Carpinteria, CA). The SpectraMax M5 microplate reader was purchased from Molecular Devices Corp.

Signaling Proteins
The two-way ANOVA tests demonstrated that ethanol had significant effects on insulin R, IRS-1, Akt, and GSK-3β expression; NNK had significant effects on all signaling proteins except IGF-1R; and ethanol × NNK interactive effects were significant for insulin R and Akt (Table 1). Graphs, together with post hoc Tukey repeated measures tests demonstrated that ethanol significantly reduced the mean expression levels of Insulin R ( Figure 1A) and IRS-1( Figure 1C), but increased p70S6K ( Figure 2J) relative to control. The effects of NNK and ethanol + NNK were largely similar in that both significantly reduced IRS-1 expression relative to control ( Figure 1C), and increased Akt ( Figure 2A) and GSK-3β ( Figure 2D) but decreased PRAS40 ( Figure 2G) relative to both control and ethanol groups. NNK and ethanol + NNK effects were distinguished by the significantly higher levels of insulin R expression in the ethanol + NNK group relative to the other three groups ( Figure 1A), and lower levels of Akt ( Figure 2A) and GSK-3β ( Figure 2D) in samples from ethanol + NNK treated relative to NNK only. There were no significant inter-group differences with respect to IGF-1R expression ( Figure 1B).

Relative Phosphorylation of Signaling Proteins
With regard to the relative levels of phosphorylation (p/T), ethanol had significant effects on the pYpY1162/1163 -Insulin R/total Insulin R and pTpS421/424 -p70S6K/total p70S6K. NNK had significant effects on pYpY1162/1163 -Insulin R/total Insulin R, pS473 -Akt/total Akt, pS9 -GSK-3β/total GSK-3β, pTpS421/424 -p70S6K/total p70S6K, and pT246 -PRAS40/total PRAS40. Ethanol × NNK interactive effects were significant only with respect to pTpS421/424 -p70S6K/total p70S6K. The graphs and post hoc tests demonstrated progressive declines in the mean levels of pYpY1162/1163 -Insulin R/total Insulin R from control to ethanol, then NNK, and finally ethanol + NNK ( Figure 1G). In addition, the mean levels of pS473 -Akt/total Akt ( Figure 2C and pT246 -PRAS40/total PRAS40 ( Figure 2I) were significantly lower in the NNK and ethanol + NNK groups relative to control and ethanol, and the levels of pTpS421/424 -p70S6K/total p70S6K ( Figure 2L) were significantly reduced in all three experimental groups relative to control. The slightly increased mean level of pS9 -GSK-3β/total GSK-3β in the ethanol group rendered the differences from NNK and ethanol + NNK statistically significant ( Figure 2F). Finally, there were no significant treatment effects on the levels of pYpY1135/1136 -IGF-1R/total IGF-1R ( Figure 1H) or pS312 -IRS-1/total IRS-1 ( Figure 1I). Overall, most of the inhibitory effects on both proximal and distal components of the insulin/IGF signaling network were driven by NNK, with or without coexposure to ethanol. The main inhibitory effects of ethanol were on insulin receptor expression and tyrosine phosphorylation, IRS-1 expression, and relative phosphorylation of p70S6K.
Two-way ANOVA tests revealed significant ethanol effects on the expression of MAG-1 and a trend effect on ASPH-A85E6, and significant NNK effects on the expression of all proteins measured except Hu and GFAP, and the calculate pTau/Tau ratio ( Table 2). Significant ethanol × NNK interactive effects were detected for MAG-1, GAPDH, and HNE, while a trend effect was detected for GFAP. The graphs in Figures 3 and 4 illustrate specific effects of the various exposures on protein expression. Hu was similarly expressed in all groups ( Figure 3A), whereas MAG-1 was significantly reduced in all experimental groups relative to control ( Figure 3B). Furthermore, MAG-1 expression, a marker of mature oligodendrocyte function, was significantly lower in the NNK and ethanol + NNK temporal lobe samples than in the ethanol-only group. GFAP, which reflects astrocyte function, was significantly reduced in the ethanol + NNK group relative to the ethanol-and NNK-only groups ( Figure 3C).
ChAT ( Figure 3D) and AChE ( Figure 3E), which regulate cholinergic homeostasis, were similarly reduced by NNK and ethanol + NNK exposures, rendering the differences from control statistically significant. GAPDH expression was significantly increased in both NNK and ethanol + NNK groups relative to control and ethanol-only treatment ( Figure 3F). The effects of ethanol, NNK, and ethanol + NNK exposures on Tau ( Figure 4A), pTau ( Figure  4B), ubiquitin ( Figure 4C), ASPH-A85E6 ( Figure 4E) and ASPH-A85G6 ( Figure 4F) expression were thematically similar in that ethanol had minimal effect relative to control, while NNK and ethanol + NNK reduced protein expression relative to both control and/or ethanol-only treatment. The calculated pTau/Tau ratios did not differ among the groups (Table 2) because the pTau levels were mainly driven by Tau protein expression rather than differential alterations in pTau. Regarding both HNE ( Figure 4D) and ASPHA85E6 ( Figure  4E), the inhibitory effects of ethanol + NNK were less than for NNK only, rendering the differences from control not statistically significant.

Early Postnatal Ethanol and NNK Exposure Model
This study examines long-term effects of early postnatal ethanol and NNK exposures on insulin and IGF-1 signaling through Akt pathways in adolescent rat temporal lobes. The experiment was designed to mimic binge drinking and smoking in the third trimester of human pregnancy. Our working hypothesis was that low-dose NNK exposures, which occur with first-or secondhand smoking, could mediate long-term impairments in brain insulin/ IGF-1 signaling through Akt pathways, and thereby cause phenotypic effects that overlap with FASD.

Ethanol and NNK Effects on Temporal Lobe Insulin/IGF-1/IRS-1 Signaling
The major findings were that: 1) ethanol and NNK independently altered the expression of proteins and phospho-proteins that mediate upstream and downstream components of the insulin R/IRS-1/Akt pathway, but had no significant effect on IGF-1R signaling; and 2) NNK, with or without ethanol co-exposure, was the main driver of impaired signaling through Akt networks that support cell survival, plasticity, and metabolism. In essence, NNK's effects were highly significant through most of the downstream steps; whereas ethanol's adverse effects were more limited its upregulation of p70S6K and inhibition of its relative phosphorylation. The finding that both ethanol and NNK inhibited pYpY1162/1163 -Insulin R expression is evidence that either alcohol or tobacco smoke exposures early in development can lead to sustained impairment of insulin signaling in adolescent brains, corresponding with previously reported effects in experimental FASD [27,48,49]. This concept is reinforced by potentially additive effects of dual exposures in which the temporal lobe levels of pYpY1162/1163 -Insulin R were lowest among the groups, despite paradoxically increased insulin R expression. The absence of ethanol and NNK effects on IGF-1R and pYpY1135/1136 -IGF-1R is discordant with previous findings [27,48,49]; however, the differences could be structure-dependent since the previous work focused on the cerebellum rather than the temporal lobe.
The greater reduction in IRS-1 protein expression in the ethanol + NNK compared with either ethanol or NNK suggests that the adverse effects of the dual exposures were additive. However, the corresponding reductions in S312 -IRS-1 in all 3 experimental groups parallel declines in IRS-1 protein, and since S312 phosphorylation of IRS-1 is inhibitory, it is unlikely that the decreases in downstream Akt signaling were not due to disruption of IRS-1 phosphorylation, and instead were mediated by decreased levels of IRS-1 protein.
In contrast to previous work in which chronic prenatal or early postnatal binge ethanol exposures were shown to have striking inhibitory effects on Akt, GSK-3β, and PRAS40 phosphorylation in the cerebellum [27,48,49], no such responses to ethanol occurred in the temporal lobe. Instead, the main downstream effects of ethanol were to increase p70S6K protein while substantially inhibiting its relative levels of phosphorylation. p70S6K, which is downstream of Akt and connected through the mammalian target of rapamycin (mTOR) pathway, promotes protein synthesis. In the brain, mTOR/p70S6K mediates brain-derived neurotrophic factor-induced protein synthesis and neuroplasticity (50), and therefore ethanol inhibition of p70S6K activation in the temporal lobe could lead to sustained impairment of neuronal plasticity required for learning and memory. NNK, with or without ethanol co-exposures, broadly inhibited Akt pathway signaling relative to control and/or ethanol exposure. With regard to Akt and GSK-3β, the NNKassociated increases in protein may have been compensatory. However, due to the absence of corresponding increases in protein phosphorylation, the relative levels of pS473 -Akt and S9 -GSK-3β were reduced. (Note that S9 phosphorylation of GSK-3β inhibits the kinase activity). In addition, NNK and ethanol + NNK significantly inhibited expression of PRAS40, pT246 -PRAS40, p70S6K, and pTpS421/424 -p70S6K, causing their relative levels to also be reduced. In essence, the net long-term effects of early postnatal NNK exposures were to inhibit virtually the entire insulin signaling pathway from receptor through downstream Akt networks that support neuronal survival, energy metabolism, protein synthesis, and plasticity. In these respects, early postnatal NNK effects on the temporal lobe mimic the longterm effects of binge ethanol exposures on the cerebellum [27,48]. Furthermore, the findings suggest that the impairments in signaling were mainly driven by NNK, since there were virtually no additive effects of the dual exposures.

Differential effects of ethanol and NNK on neuronal and glial protein expression
Ethanol, NNK, and ethanol + NNK exposures all significantly reduced temporal lobe levels of MAG-1 expression relative to control, although the effects of NNK and ethanol + NNK were more pronounced than ethanol's. MAG-1, a glycoprotein expressed in oligodendrocytes, is responsible for facilitating cell-cell interactions between neuronal and myelinating cells. Ethanol's inhibitory effects on white matter development are wellestablished and have been linked to impairments in oligodendrocyte myelin-associated gene/ protein expression [1]. The finding that developmental exposures to NNK can also reduce MAG-1 expression is novel and supports the hypothesis that alcohol and tobacco smoke exposures can both contribute to white matter hypotrophy and reduced myelination in adolescent brains. In contrast, there were no significant differences in the expression levels of Hu, a marker of neurons, or GFAP, the main intermediate filament protein of mature astrocytes, in the experimental groups relative to control. These findings highlight the selective targeting of oligodendrocytes by ethanol and NNK.
Acetylcholine, one of the major neurotransmitters utilized for neuronal plasticity in the brain, is regulated by ChAT for biosynthesis, and AChE for degradation. The absence of ethanol effects on ChAT and AChE is discordant with previous findings in studies of the cerebellum [5,8,14]. We speculate that rapidly proliferating, migrating and differentiating neurons in early postnatal cerebella are more vulnerable as targets of ethanol neurotoxicity than post-mitotic temporal lobe neurons. On the other hand, the findings that ChAT and/or AChE expression were reduced by developmental exposures to NNK suggest that postmitotic temporal lobe neurons are susceptible to the delayed neurotoxic effects of NNK. Reduced expression of ChAT correlates with impaired insulin signaling [8]. Inhibition of AChE expression can be mediated by oxidative stress [51][52][53][54], such as that caused by the impairments of insulin signaling through Akt with increased activation of GSK-3β, as occurred in brains from NNK-exposed rats. Inhibition of AChE can be sufficient to cause cytoskeletal collapse and neurodegeneration [55]. Together, these findings suggest that NNK and therefore tobacco smoke exposures in the early postnatal period (equivalent to 3 rd trimester of human pregnancy) can impair temporal lobe cholinergic function which is needed for neuronal plasticity, learning and memory.
Further studies showed that NNK and ethanol + NNK similarly reduced tau, p-tau, ubiquitin, ASPH-A85G6 and ASPH-A85E6 protein expression relative to control and ethanol exposures. These responses were driven by NNK since ethanol had no independent or additive effects. Tau is a major neuronal cytoskeletal protein whose phosphorylation state is critical for translocation from the perikarya into neurites for establishing and maintaining synaptic connections. Therefore, NNK's Inhibition of Tau and p-Tau expression could reflect retraction or degeneration of axons, collapse of growth cone, and synaptic disconnection [56]. Since tau expression and phosphorylation are regulated by insulin and IGF-1 signaling through Akt and GSK-3β [57][58][59], it is not surprising that these proteins were significantly reduced by NNK exposures, given the prominent inhibition of insulin Rand Akt phosphorylation. The finding that the relative levels of pTau (pTau/Tau) were not significantly reduced vis-à-vis significant reductions Tau and pTau following NNK exposure indicates that the main effect of NNK was to inhibit Tau expression. The similarly reduced levels of pTau are best explained by the lower levels of protein rather than impaired signaling and kinase activation via GSK-3β. On the other hand, in ethanol-exposed temporal lobes, the relatively normal levels of tau and pTau could be explained by preservation of signaling through Akt and GSK-3β. The NNK associated reductions in ubiquitin could reflect deficits in the ubiquitin-proteasome system. A similar response occurs following chronic ethanol exposure [60,61]. Deficits in the ubiquitin-proteasome pathway could lead to increased oxidative and endoplasmic reticulum stress due to activation of the unfolded protein response [62,63].
For ASPH, we used the A85G6 monoclonal antibody that binds to the C-terminal region of ASPH which contains a catalytic domain, and A85E6, that binds to the N-terminal Humbughomologous region of ASPH [34,40,64]. The catalytic domain of ASPH is required to promote cell motility [35][36][37]65,66] and neuronal plasticity [35][36][37][38][39][40]67]. Humbug regulates calcium sequestration in the ER (68). ASPH expression and function are regulated by insulin/IGF-1 signaling through IRS-1 and Akt [35,40,67]. Inhibition of ASPH perturbs cell motility and adhesion [36,39,69], and in the case of FASD, ethanol's inhibitory effects on ASPH expression correlate with impairments in cerebellar neuronal migration and motor dysfunction [34,64]. The findings herein demonstrate that early post-natal exposures to NNK significantly inhibit temporal lobe expression of ASPH and Humbug, correlating with reduced activation of Akt. In contrast, ethanol had no significant effect on these proteins, corresponding with the preservation of signaling through Akt and GSK-3β in the temporal lobe.